Albright hereditary osteodystrophy (AHO) is an autosomal dominant disorder characterized by short stature, obesity, subcutaneous ossifications, and brachydactyly. Some family members have AHO features only (pseudopseudohypoparathyroidism, PPHP) while others have AHO in association with resistance to multiple hormones which activate Gs-coupled receptors (pseudohypoparathyroidism type Ia, PHP Ia). AHO/PHPIa is caused by heterozygous Gs-alpha null mutations and most affected patients have a 50% deficiency in Gs-alpha subunit function and/or expression in peripheral tissues (both PHP Ia and PPHP). Gs-alpha is a ubiquitously expressed G protein alpha-subunit that is required for the cyclic AMP response to hormones and other extracellular signals. We have shown in mice and more recently in humans that Gs-alpha is imprinted in a tissue-specific manner. In some hormone target tissues Gs-alpha is expressed primarily from the maternal allele, and therefore maternal inheritance of Gs-alpha mutations results in PHP Ia while paternal inheritance of these same mutations leads to PPHP. More recently GNAS/Gnas has been shown to be a very complicated gene with multiple imprinted gene products generated by several alternative promoters and first exons. NESP55 is a chromogranin-like protein that is maternally expressed while XL-alpha-s is a paternally expressed Gs-alpha isoform with a long amino-terminal extension. Both are primarily expressed in neuroendocrine tissues. We have shown that NESP imprinting is not established until postimplantation development. Just upstream of the XL-alpha-s promoter is the promoter for a paternally-expressed antisense transcript (NESPAS) that traverses the NESP promoter from the opposite direction. We identified another alternative first exon (exon 1A) that generates paternally expressed untranslated mRNAs and that is a maternal germline imprint mark. We have shown that this region has allele-specific differences in histone methylation. We also have shown that the Gsa promoter and first exon also has allele-specific differences in histone methylation which correlates to its tissue-specific imprinting, even though this region does not undergo DNA methylation. We have shown that PHP Ib (parathyroid hormone resistance in the absence of AHO) is virtually always associated with loss of maternal exon 1A imprinting. A detailed analysis of GNAS imprinting in PHP Ib patients showed that familial cases tend to only have abnormal exon 1A imprinting associated with a deletion mutation within a closely-linked gene, while sporadic cases often have additional imprinting defects involving NESP and XL-alpha-s. In some patients, the imprinting of the XL-alpha-s promoter and its first exon is discordant. We have examined Gnas methylation in mice which do not establish maternal germline imprints (dnmt3L-/-) and show that the whole Gnas locus develops a paternal methylation pattern on both alleles, indicating that imprinting of the whole locus depends on maternal germline imprints. We have also generated exon 1A knockout mice, and show that this region is not required for Nesp and XL-alpha-s imprinting, but is required on the paternal allele for tissue-specific Gs-alpha imprinting. We originally showed that mice that inherit a Gnas exon 2 insertion paternally are leaner than normal and are hypermetabolic and hyperactive, while mice which inherit the mutation maternally become obese, hypometabolic, and hypoactive. Detailed metabolic studies have shown the paternal exon 2 knockout mice to have increased adiponectin and resistin expression in adipose tissue, increased whole body lipid metabolism, and increased insulin sensitivity in adipose tissue, muscle, and liver. We have created a new mouse line with flox sites around Gs-alpha exon 1, and with this have made mice with Gs-alpha specific deficiency. Mice with the knockout on the paternal allele have an opposite metabolic phenotype to those with the exon 2 insertion (obesity, insulin resistance, poor lipid clearance). We believe that the paternal exon 2 phenotype is caused by loss of XL-alpha-s and that this Gs-alpha isoform may play a role in regulating sympathetic activity in mice such that loss of this protein leads to a sympathetic hyperactivity. We have also generated tissue-specific Gs-alpha knockouts in various metabolically active tissues and bone. Preliminary findings show Gs-alpha to have major roles in growth plate development, osteoblast function, insulin action, adipocyte function, and muscle glucose uptake.